Our primary goal is the development of useful synthetic glycopeptide mimics that effectively represent cell surface O-glycopeptides initiated as alpha-linked: O-GalNAc and O-Man Ser/Thr. These mimics or surrogates will provide metabolic stability to enhance bioavailability and are expected to have altered binding affinities to immune surveillance molecules to help break inherent immunotolerance by eliciting T-cell help. The (R-CHOH)-GalNAc-a-Ser/ThrF3 will be prepared as mimics of Tn antigen, O-GalNAc-a-Ser/Thr, and will be incorporated into two biomedically relevant peptides. This improved mimetic design, based on NMR and x-ray crystallographic studies, should have potential therapeutic applications in bioassays, immunotherapy, and antineoplastic activity. The (CH2)-Man/ManF-a-Ser will serve as a robust mimic of hypoglycosylated a-dystroglycan (a-DG) and other O-mannosyl proteins in order to raise glycan-specific antibodies. The generation of new O-Man specific mAbs would be useful for isolation and identification of glycoproteins known to be implicated in not only a range of congenital muscular dystrophies, but also in tumor metastasis and in autoimmune diseases such as multiple sclerosis.
The initial design and syntheses of the desired mimetics are proposed (Aims 1 and 2). After establishing the synthetic routes, we will adjust the protecting groups to provide samples for the NMR analysis and for solid-phase peptide synthesis to explore the conformational (Aim 3), and immunological and tumor inhibition studies (Aim 4).
Aim 1. Design and Syntheses of (R-CHOH)-GalNAc-a-Ser/Thr(F3) with Comparative Analogs:
The all-carbon skeleton, from the Ser/Thr-carbonyl to C6 of the glycoside, of mimics 1 and 2 will be constructed using metathesis chemistry and Pd-catalyzed cyclization. A C-vinyl glycoside with an unprotected hydroxyl at C2 serves as a branching point for synthetic routes. The C2-Gal functionality (hydroxyl for 2 and oxime for 1) will provide a handle for delivery of the pendant hydroxyl group (shown in red) onto the linking chain establishing the R-configuration at C1’—a feature designed to promote good conformational mimicry of O-Tn antigen.
Aim 2. Synthesis (CH2)-Man/Man2F-a-Ser:
As in Aim 1, metathesis and Pd-catalyzed cyclization will afford a gluco-configured derivative. The C2-hydroxyl will be inverted to yield the mannose target 3 and the 2-deoxy-2-fluoromannose 3F.
Aim 3. Conformational Study of the Effects of the Glycopeptide Analogs by NMR Analysis.
O-Glycopeptide structural studies, specifically related to Tn antigen, have highlighted preferred conformations and forces responsible for orienting the glycan moiety with respect to the peptide backbone, and vice versa. The intramolecular interaction of the glycan back toward the peptide is critical for the expected intermolecular interactions of the glycan face presented away from the peptide. Our synthetic (R-CHOH)-mimics 1, and comparative analogs 2, will be analyzed by NMR and molecular dynamics simulations for comparison of the conformational changes affected by altering the natural O-glycosyl linkages. Incorporation of these into simple peptide models and biomedically-relevant peptides will be carried out to provide samples for the NMR studies.
Aim 4. Incorporation of Mimics into MUC1 and a-DG for Immunological Studies and APF for Tumor Inhibition Studies:
The glycopeptides of interest that will be modified are: 1. MUC 1, a mucin transmembrane glycoprotein that includes an extracellular 20-mer tandem repeat displaying tumor-associated carbohydrate antigens, such as Tn antigen, which are typically masked in normal cells but exposed during carcinogenesis; 2. a peptide growth factor, known as antiproliferative factor or APF, which is a glycosylated nonapeptide isolated from bladder cells of patients with interstitial cystitis and found to inhibit bladder tumor cell growth; and 3. three 9-10-mers from a-DG known as common sites for mannosylation. The mimics from Aims 1 and 2 will be incorporated in these glycopeptides using solid-phase peptide synthesis and supplied to collaborators for immunological and inhibition studies.